Obatoclax interacts synergistically with the irreversible proteasome inhibitor carfilzomib in GC- and ABC-DLBCL cells in vitro and in vivo

Abstract

Interactions between the irreversible proteasome inhibitor carfilzomib and the pan-BH3 mimetic obatoclax were examined in germinal center (GC)- and activated B-cell-diffuse large B-cell lymphoma (ABC-DLBCL) cells. Cotreatment with minimally toxic concentrations of carfilzomib (i.e., 2-6 nmol/L) and subtoxic concentrations of obatoclax (0.05-2.0 μmol/L) synergistically increased apoptosis in multiple DLBCL cell lines and increased lethality toward primary human DLBCL but not normal CD34(+) cells. Synergistic interactions were associated with sharp increases in caspase-3 activation, PARP cleavage, p-JNK induction, upregulation of Noxa, and AKT dephosphorylation. Combined treatment also diminished carfilzomib-mediated Mcl-1 upregulation whereas immunoprecipitation analysis revealed reduced associations between Bak and Mcl-1/Bcl-xL and Bim and Mcl-1. The carfilzomib/obatoclax regimen triggered translocation, conformational change, and dimerization of Bax and activation of Bak. Genetic interruption of c-jun-NH(2)-kinase (JNK) and Noxa by short hairpin RNA knockdown, ectopic Mcl-1 expression, or enforced activation of AKT significantly attenuated carfilzomib/obatoclax-mediated apoptosis. Notably, coadministration of carfilzomib/obatoclax sharply increased apoptosis in multiple bortezomib-resistant DLBCL models. Finally, in vivo administration of carfilzomib and obatoclax to mice inoculated with SUDHL4 cells substantially suppressed tumor growth, activated JNK, inactivated AKT, and increased survival compared with the effects of single-agent treatment. Together, these findings argue that a strategy combining carfilzomib and obatoclax warrants attention in DLBCL.

Figure 1. Carfilzomib/obatoclax co-treatment synergistically induces cell death in DLBCL lymphoma lines and primary lymphoma cells but not in normal cells

(A). SUDHL16 cells were treated (48 hr) with carfilzomib (CFZ) (2.0 – 4.0 nM) ± obatoclax (obato;150–250 nM), after which apoptosis was monitored by 7AAD.. B. SUDHL16 cells were treated (48 hr) with 100–300 nM obatoclax ± carfilzomib (2.0–3.0 nM), followed by 7AAD staining C. Time course of SUDHL16 cell death (7AAD) following obatoclax/carfilzomib exposure. (D) Synergistic interactions between carfilzomib and obatoclax at various concentrations in SHDHL16 cells were tested using Laska’s model-free test for synergy as in Methods. Synergism was observed for all 8 combinations tested with P values ranging from < 0.001 to 0.0027. (E) Combination Index (C.I.) values were determined using CalcuSyn software employing fixed drug ratios of carfilzomib and obatoclax (1:80) in SUDHL16 cells (F) OCI-LY10 and OCI-LY3 cells were treated (48 hr) with carfilzomib (5.0 and 4.0 nM respectively) ± obatoclax (50 and 75nM respectively); cell death was monitored by 7AAD/DiOC6 staining. (G) Two primary human DLBCL (one ABC and other GC sub-type) specimen was isolated and treated with carfilzomib (2 nM and 100 nM for ABC and GC sub types respectively) ± obatoclax (75nM and 125nM for ABC and GC sub types respectively) for 14 h, after which cell death was monitored by 7AAD staining and normalized to controls. Viability of untreated primary specimens was 80% (H) CD34⁺ cells were exposed (48 hr) to carfilzomib (10–20 nM) ± obatoclax (500 nM). Cell death was monitored by 7AAD staining and normalized to controls. For all studies, values = means for 3 experiments performed in triplicate ± S.D. For A–C, F, G * = values significantly greater than those for carfilzomib or obatoclax treatment alone; P < 0.01.

Figure 2. Carfilzomib/obatoclax co-exposure activates JNK, inactivates AKT, up-regulates Noxa, and induces γH2A.X in DLBCL cells

SUDHL16 cells were treated (24 hrs) with carfilzomib (2.5 nM) ± obatoclax (200nM). (A-D) Expression of the indicated proteins was determined by Western blotting (E), SUDHL16 cells were treated with 2.5nM of carfilzomib and Mcl-1 determined by Western blotting. Each lane was loaded with 20 μg of protein; blots were stripped and re-probed with antibodies directed against tubulin to ensure equivalent loading and transfer. Results are representative of three separate experiments (F) OCI-Ly10 cells were treated with carfilzomib (4.0 nM) ± obatoclax (50nM) for 24 hrs and expression of the indicated proteins determined by Western blotting

Figure 3. Genetic interruption of JNK and AKT significantly diminishes carfilzomib/obatoclax lethality

(A) SUDHL16 - JNK shRNA or scrambled sequence vectors were exposed (48 hr) to carfilzomib (2.5nM) + obatoclax (150nM), after which, cell death was monitored by 7AAD.. Inset: expression of JNK protein in SUDHL16-scrambled sequence and shJNK clones. (B) Following 24 hr treatment as in (A) protein was monitored by Western blot (C) SUDHL16 cells were stably transfected with constitutively active (myristolated) AKT constructs (AKT cl.3 and 5) or empty vector (pUSE) and exposed (48 hr) to carfilzomib (3.0nM) + obatoclax (150nM), after which cell death was monitored by 7-AAD. Inset: Western blots showing expression of AKT and p-GSK-3α/β in empty vector control and AKT clones. (D) Cells were treated as described above in (C) for 24 hrs, after which Western blot analysis was performed to monitor expression of the indicated proteins. For A and C * = significantly less than values obtained for carfilzomib + obatoclax treatment in SUDHL16 scrambled sequence or SUDHL16 expressing pUSE cells; P < 0.05.

Figure 4. Noxa up-regulation and Mcl-1 down-regulation play functional roles in potentiating carfilzomib/obatoclax lethality in DLBCL cells

(A) SUDHL4 cells were transiently transfected with a Noxa construct or an empty vector (pRFP-C-RS) and exposed (48 hr) to carfilzomib (1.5 nM) + obatoclax (1.0 μM), after which. Cell death was monitored by 7-AAD. Inset: Western blots showing expression of Noxa in empty vector control and Noxa transfected cells following carfilzomib (1.5 nM) + obatoclax (1.0μM) treatment. (B) Cells were treated as in (A) for 24 hrs, after which expression of the indicated proteins was monitored. (C) SUDHL4 cells were transiently transfected with a Mcl-1 construct or an empty vector (pcDNA3.1) and exposed (48 hr) to carfilzomib (2.0 nM) + obatoclax (1.0 μM) for 48 hrs. Cell death was monitored by 7-AAD. Inset: Western blots showing expression of Mcl-1 in empty vector control and transiently transfected cells. (D) Cells were treated as in (C) for 24 hrs, after which Western blot analysis was performed to monitor protein expression. Immunoprecipitation was employed to characterize the association between Bim and Mcl-1. For A and C * = significantly less than values obtained for carfilzomib + obatoclax treatment in SUDHL16-scrambled sequence or SUDHL16 expressing pcDNA3.1 control cells; P < 0.05.

Figure 5. The CFZ/obatoclax regimen potently induces apoptosis in bortezomib-resistant SUDHL16-10BR, and OCY-LY10-40BR cells

(A) SUDHL16-10BR and OCI-LY10-40BR cells were treated 48 hr) with minimally toxic concentrations of carfilzomib and obatoclax. Concentrations were as follows: SUDHL16-10BR - carfilzomib (5 nM) ± obatoclax (250 nM), OCI-LY10-40BR - carfilzomib (20 nM) ± Obatoclax (100 nM); Cell death was monitored by 7AAD. (B) Median Dose Effect analysis yielded. Combination Index (C.I.) values < 1.0 denoting synergistic interactions. C. SUDHL16-10BR cells were exposed (24 hr) to carfilzomib and obatoclax as in (A), after which Western blot analysis was performed.. D. SUDHL16-10BR and OCI-LY10-40BR cells were treated with carfilzomib and obatoclax for 24 hrs as in (A) after which, associations between Mcl-1 and Bak and BIM, Bcl-xL with Bak were characterized by immunoprecipitation followed by Western blotting.

Figure 6

Obatoclax markedly potentiates CFZ induced tumor growth suppression, survival, JNK activation, and AKT dephosphorylation in an in vivo SUDHL4 xenograft model NIH-III nude mice were injected in the flank with (A) 10 x10⁶ SUDHL4-luciferase cells and treated with carfilzomib (2.0mg/kg) ± obatoclax (3.0mg/kg) as described in Methods. Tumor volumes were measured twice weekly and mean tumor volumes were plotted against days of treatment. (B) Tumor progression was also monitored using an IVIS bioimager. (C). Survival curve of individual groups (control-blue line, carfilzomib- green line, obatoclax - yellow line, , carfilzomib + obatoclax - purple line) was evaluated from the first day of treatment until death using Kaplan–Meier curves (*, P < 0.05). (D) Tumor samples were lysed with lysis buffer followed by sonication. Western blotting was performed to monitor expression of the indicated proteins.